Patent GB-817729-A (GB)
Title: A new antibiotic p.a. 371
Filed: 1955-10-19
Abstract: An antibiotic designated P.A. 371 and having six components alpha, alpha-prime, beta, gamma, delta and epsilon, is produced by cultivating Streptomyces ATCC 11926 (described) or a mutant or variant thereof, in an aqueous nutrient medium under aerobic conditions, preferably submerged aerobic conditions at a temperature of 24 DEG  to 30 DEG  C. for 1 to 4 days.  The medium contains a source of (a) carbon such as sugars, starch, glycerol and corn meal, (b) nitrogen such as urea, casein, soybean meal, peanut meal, wheat gluten, cottonseed meal, lactalbumin, tryptone, and enzymatic digest of casein, (c) inorganic salts such as ammonium salts, e.g. (NH4)2SO4, NH4Cl and ammonium phosphates, sodium chloride, potassium phosphate, magnesium sulphate, alkali metal nitrates and trace metals (d) growth substances such as distillers solubles yeast extract, molasses fermentation residues, and (e) buffer such as calcium carbonate. The antibiotic is isolated by (a) solvent extraction, (b) adsorption on an ion-exchange resin, or (c) column chromatography. Crude PA 371 is obtained by extracting the filtered broth at pH5 to 8 with a water-immiscible partially polar organic solvent (ethyl acetate, methylisobutyl ketone, methylethyl ketone or methylene chloride), concentrating the extract and adding hexane. Crude hydrochlorides are produced by treating the solvent concentrate with hydrogen chloride.  Crude hydrochlorides treated with picric acid to form picrates and digested with acetone leave a residue of P.A. 371 beta picrate.  P.A. 371 Alpha picrate may be crystallized from the acetone solution.  Where the crude is high in the alpha component, the alpha picrate may be obtained by treating the crude P.A. 371 hydrochlorides with picric acid in methanol.  P.A. 371 beta sulphate is less soluble than sulphates of other components and may be separated by adding triethylamine sulphate to a solution of the mixed components.  P.A. 371 beta base may be precipitated by adding a phosphate buffer of pH 7.0 to a water solution of the mixed hydrochlorides.  Owing to the acid addition salts of the beta component being less soluble than those of the other components P.A. 371 beta 5-nitrosalicylate and beta naphthalene sulphonate are prepared by adding the appropriate organic acid to an aqueous solution of the mixed components.  P.A. 371 Alpha prime and P.A. 371 gamma are obtained as acetates by crystallizing a methylene chloride extract of a neutralized acetic acid wash obtained from a methylisobutyl ketone extract of a filtered broth.  P.A. 371 delta and P.A. 371 epsilon occur in small amounts and are separated by paper chromatography or countercurrent distribution, e.g. using toluenethyl acetate-methanol water.  Mixed components may be isolated from the filtered broth by adsorption or a carboxylic acid ion exchange resin in the hydrogen form and elution with an aliphatic acid, e.g. acetic, alcohol, e.g. methanol, and water mixture.  P.A. 371 is active against <PICT:0817729/IV (b)/1> <PICT:0817729/IV (b)/2> <PICT:0817729/IV (b)/3> <PICT:0817729/IV (b)/4> <PICT:0817729/IV (b)/5> <PICT:0817729/IV (b)/6> a variety of Gram-positive microorganisms and mycobacteria, the activity of the components varying between themselves.  It is active against streptomycin and isoniazid resistant microorganisms.  P.A. 371 is a yellow amphoteric solid, red in solutions of alkaline pH, yellow at acid pH.  It is soluble in water butanol, ethyl acetate, methyl ethyl ketone and insoluble in petroleum ether.  The components have the following properties: <TABS:0817729/IV (b)/1> In the above table, the balance of the constituents of the compounds is oxygen.  The components have similar ultra-violet absorption curves showing maxima at 228 mm  and 257 mm , a shoulder at 290 and a diffuse maximum centring at 425 mm . The characteristics of the infra-red absorption spectra are shown in Figs. 1 to 6.  The antibiotics are used in medicinal compositions. Specification 690,381 is referred to.ALSO:Antibiotic PA371 having six components alpha, alpha prime, beta, gamma, delta and epsilon, produced by cultivating Streptomyces ATCC11926, or a mutant or variant thereof (see Group IV (b)), which is active against gram positive microorganisms and mycobacteria including streptomycin-resistant strains thereof, is incorporated in pharmaceutical and veterinary compositions.  It may be used in the form of a powder, tablet, capsule, or suspension containing wetting agents.